Abstract

Cuticle, the outermost layer covering all primary aerial surfaces of vascular land plants, is essential for the protection and normal development of the plant and has as main components cutin and wax. Although numerous genes are involved in the biosynthesis and regulation of cuticular components, only few have a known function. To contribute to the understanding of regulatory mechanisms involved in cuticle development, two approaches have been taken in this project. These involved on the one hand mapping and characterizing a locus involved in wax regulation and metabolism and on the other hand, identifying putative interactors for three proteins involved in cuticular pathways. The first approach concerns the mapping and characterization of eceriferum13 (cer13) mutant Arabidopsis thaliana. Obtained in Ler-0 background by fast neutron treatment, the cer13 mutant has less epicuticular wax and its semi-dwarf phenotype, as well as its responsiveness to photoperiod indicate a possible implication in regulatory mechanisms. After fine mapping to a region of 46 candidate genes, RESURRECTION1 (RST1, AT3G27670) was selected as candidate for CER13, due to the fact that its mutation causes altered epicuticular wax phenotype. Sequence analysis of RST1, encoding a putative transmembrane protein of unknown function, revealed a 950 bp deletion resulting in a frame shift and a premature STOP codon in CER13 cDNA. The allelism test performed by crossing cer13 to rst1-3 was positive, indicating a single locus. To investigate the mechanism responsible for the reduced wax levels in cer13, we performed a wholegenome ATH1 microarray analysis. Previously, the biochemical pattern of wax composition was found to be highly similar between cer13, cer3 and cer7. Interestingly, CER3 is down-regulated in both cer13 and cer7 transcriptomes, indicating that the three genes could be involved in the same pathway. We also compared cer13-misregulated genes to a MASTA (MicroArray overlap Search Tool and Analysis) database of more than 600 microarray datasets. This analysis suggested that CER13 provides a link between wax biosynthesis and salicylic acidmediated signaling, as well as similar expression patterns to cold and drought stress. The second approach undertaken in this study consists in using three cuticular genes as baits in a Y2H cDNA library screening. All three genes that were selected for this screening are involved in the cuticle development in an unusual manner. The putative interactors found for the subtilisin-like serine protease ALE1, which is required for the proper formation of epidermis and cuticle around the embryo(Tanaka et al., 2001), are subunits a and b of the CSN5, a component of the COP9 signalosome. The putative acyl transferase CER2 is particular in that it localizes to the nucleus. A porin and two immunophilins were identified as putative interactors. The PALMITOYL PROTEIN THIOESTERASE (PPT) is a gene up-regulated in three independent cuticular mutants and an ATP-dependent helicase as well as an ADPribosilation factor were isolated as putative interactors. These results highlight the complexity of the regulatory mechanisms behind the formation and the function of the cuticle and contribute to the understanding of plant defenses.